Inactivation by Hg(2+) and methylmercury of the glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Prediction of the involvement of a CXXC motif by homology modelling

The effect of HgCl(2), methylmercury and mersalyl on the glutamine/amino acid (ASCT2) transporter reconstituted in liposomes has been studied. Mercuric compounds externally added to the proteoliposomes, inhibited the glutamine/glutamine antiport catalyzed by the reconstituted transporter. Similar effects were observed by pre-treating the proteoliposomes with the mercurials and then removing unreacted compounds before the transport assay. The inhibition was reversed by DTE, cysteine and N-acetyl-cysteine but not by S-carboxymethyl-cysteine. The data demonstrated that the inhibition was due to covalent reaction of mercuric compounds with Cys residue(s) of the transporter. The IC(50) of the transporter for HgCl(2), methylmercury and mersalyl, were 1.4 +/- 0.10, 2.4 +/- 0.16 or 3.1 +/- 0.19 mu M, respectively. Kinetic studies of the inhibition showed that the reagents behaved as non-competitive inhibitor. The presence of glutamine or Na(+) during the incubation of the mercuric compounds with the proteoliposomes did not exerted any protective effect on the inhibition. None of the compounds was transported by the reconstituted transporter. A metal binding motif CXXC has been predicted as possible site of interaction of the mercuric compounds with the transporter on the basis of the homology structural model of ASCT2 obtained using the glutamate transporter homologue from Pyrococcus horikoshii as template. (C) 2010 Elsevier Inc. All rights reserved.